Enzymatic hydrolysis of starry triggerfish (Abalistes stellaris) muscle using liver proteinase from albacore tuna (Thunnus alalunga).

Proteinases from liver extract from albacore tuna (Thunnus alalunga) were used to produce protein hydrolysate from starry triggerfish (Abalistes stellaris) muscle. Hydrolysis conditions for preparing protein hydrolysate from starry triggerfish muscle were optimized. Enzyme level, reaction time and fish muscle/buffer ratio significantly affected the hydrolysis (p < 0.05). Optimum conditions for triggerfish muscle hydrolysis were 5.5 % liver extract, 40 min reaction time and fish muscle/buffer ratio of 1:3 (w/v). The freeze-dried protein hydrolysate was characterized with respect to chemical composition, amino acid composition and color. The product contained 91.73 % protein, 2.04 % lipid and 6.48 % ash. The protein hydrolysate exhibited high amount of essential amino acids (45.62 %). It was light yellow in color (L (*) = 82.94, a (*) = 0.84, b (*) = 22.83). The results indicate that the extract from liver of albacore tuna could be used to produce fish protein hydrolysate and protein hydrolysate from starry triggerfish muscle may potentially serve as a good source of desirable peptide and amino acids.

Tuna pepsin: characteristics and its use for collagen extraction from the skin of threadfin bream (Nemipterus spp.).

Pepsin from the stomach of albacore tuna, skipjack tuna, and tongol tuna was characterized. Pepsin from all tuna species showed maximal activity at pH 2.0 and 50 degrees C when hemoglobin was used as a substrate. Among the stomach extract of all species tested, that of albacore tuna showed the highest activity (40.55 units/g tissue) (P < 0.05). Substrate-Native-PAGE revealed that pepsin from albacore tuna and tongol tuna consisted of 2 isoforms, whereas pepsin from skipjack tuna had only 1 form. The activity was completely inhibited by pepstatin A, while EDTA (ethylenediaminetetraacetic acid), SBTI (soybean trypsin inhibitor), and E-64 (1-(L-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane) exhibited negligible effect. The activity was strongly inhibited by SDS (sodium dodecyl sulfate) (0.05% to 0.1%, w/v). Cysteine (5 to 50 mM) also showed an inhibitory effect in a concentration dependent manner. ATP, molybdate, NaCl, MgCl(2), and CaCl(2) had no impact on the activity. When tuna pepsin (10 units/g defatted skin) was used for collagen extraction from the skin of threadfin bream for 12 h, the yield of collagen increased by 1.84- to 2.32-fold and albacore pepsin showed the comparable extraction efficacy to porcine pepsin. The yield generally increased with increasing extraction time (P < 0.05). All collagen obtained with the aid of tuna pepsin showed similar protein patterns compared with those found in acid-solubilized collagen. Nevertheless, pepsin from skipjack tuna caused the degradation of alpha and beta components. All collagens were classified as type I with large portion of beta-chain. However, proteins with molecular weight (MW) greater than 200 kDa were abundant in acid-solubilized collagen.

Improvement of gelling properties of lizardfish mince as influenced by microbial transglutaminase and fish freshness.

The effects of microbial transglutaminase (MTGase) at different levels (0 to 0.8 units/g sample) on the properties of gels from lizardfish (Saurida undosquamis) mince set at 25 degrees C for 2 h or 40 degrees C for 30 min prior to heating at 90 degrees C for 20 min were studied. Breaking force and deformation of gels increased with increasing MTGase amount added (P<0.05). At the same MTGase level used, gels with the prior setting at 40 degrees C for 30 min showed a higher breaking force compared with those subjected to prior setting at 25 degrees C for 2 h (P<0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic study revealed that myosin heavy chain (MHC) underwent polymerization to a higher extent in the presence of MTGase. Regardless of setting condition, microstructure of gel added with MTGase was finer with a smaller void compared with that of gel without MTGase. Therefore, setting temperature affected the property of gels added with MTGase. Gel properties of mince obtained from lizardfish stored in ice for different times (0 to 10 d) with and without MTGase at a level 0.6 units/g were determined. Irrespective of MTGase addition, breaking force and deformation of all gels decreased as the storage time of lizardfish increased (P<0.05). The addition of MTGase was able to increase both breaking force and deformation of the resulting gel produced from lizardfish kept in ice for all storage times used. Therefore, both freshness and MTGase addition had the direct impact on gel properties of lizardfish mince.

Isolation and characteristics of trypsin inhibitor from the hepatopancreas of a squid (Todarodes pacificus).

Trypsin inhibitor was purified from the hepatopancreas of squid (Todarodes pacificus). The final inhibitor preparation was nearly homogeneous by SDS-PAGE with an estimated molecular weight of approximately 6300. The squid trypsin inhibitor was acid- and heat-stable, and active against trypsins from the pyloric ceca of starfish (Asterias amurensis) and saury (Cololabis saira) and porcine pancreatic trypsin. Amino acid composition of the squid trypsin inhibitor was compared with other invertebrate trypsin inhibitors. The squid trypsin inhibitor inhibited the autolysis of walleye pollock (Theragra chalcogramma) myofibrillar proteins.

Bacterial expression and characterization of starfish phospholipase A(2).

Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris--HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera.

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A2 (PLA2), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1-414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA2 protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA2 protein, suggesting the presence of multiple forms in the starfish PLA2. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in beta-wing region when aligned with the sequence of mammalian pancreatic PLA2. In phylogenetic tree, the starfish PLA2 should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and beta-wing regions may account for the specific properties of the starfish PLA2, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA2 from porcine pancreas.

Preparation of neoglycoprotein from carp myofibrillar protein and alginate oligosaccharide: improved solubility in low ionic strength medium.

Alginate oligosaccharide (AO) was conjugated with carp myofibrillar protein (Mf) by using the controlled Maillard reaction, and the change in the solubility of Mf in low ionic strength media as affected by the glycosylation was investigated. AO was prepared by degrading sodium alginate using alginate lyase, which was purified from the culture supernatant of Pseudoalteromonas elyakovii. When a lyophilized Mf and AO mixture was incubated at 40 degrees C and 65% relative humidity, the conjugation of AO was confirmed at myosin heavy chain, actin, and tropomyosin. When >30 microg/mg of AO was conjugated to Mf, the protein solubility in a low ionic strength medium was greatly improved without significant loss of available lysine. These results indicate that the conjugation with AO is a superior manner for improving the water solubility of Mf in view of its nutritional aspect.